A Stable Chimeric Fibroblast Growth Factor (FGF) Can Successfully Replace Basic FGF in Human Pluripotent Stem Cell Culture

Fibroblast growth factors (FGFs) are essential for maintaining self-renewal in human embryonic stem cells and induced pluripotent stem cells. Recombinant basic FGF (bFGF or FGF2) is conventionally used to culture pluripotent stem cells; however, because of the instability of bFGF, repeated addition of fresh bFGF into the culture medium is required in order to maintain its concentration. In this study, we demonstrate that a heat-stable chimeric variant of FGF, termed FGFC, can be successfully used for maintaining human pluripotent stem cells. FGFC is a chimeric protein composed of human FGF1 and FGF2 domains that exhibits higher thermal stability and protease resistance than do both FGF1 and FGF2. Both human embryonic stem cells and induced pluripotent stem cells were maintained in ordinary culture medium containing FGFC instead of FGF2. Comparison of cells grown in FGFC with those grown in conventional FGF2 media showed no significant differences in terms of the expression of pluripotency markers, global gene expression, karyotype, or differentiation potential in the three germ lineages. We therefore propose that FGFC may be an effective alternative to FGF2, for maintenance of human pluripotent stem cells.     

Switch from alphavbeta5 to alphavbeta6 integrin expression protects squamous cell carcinomas from anoikis

Stratified squamous epithelia express the alphavbeta5 integrin, but in squamous cell carcinomas (SCCs) there is down-regulation of alphavbeta5 and up-regulation of alphavbeta6. To investigate the significance of this finding, we transduced an alphav-negative human SCC line with retroviral vectors encoding alphav integrins. alphavbeta5-expressing cells underwent suspension-induced apoptosis (anoikis), whereas alphav-negative cells and cells expressing alphavbeta6 did not. Resistance to anoikis correlated with PKB/Akt activation in suspension, but not with changes in PTEN or p110alpha PI3 kinase levels. Anoikis was induced in parental and alphavbeta6-expressing cells by inhibiting PI3 kinase. Conversely, activation of Akt or inhibition of caspases in alphavbeta5-expressing cells suppressed anoikis. Caspase inhibition resulted in increased phosphoAkt, placing caspase activation upstream of decreased Akt activation. Anoikis required the cytoplasmic domain of beta5 and was independent of the death receptor pathway. These results suggest that down-regulation of alphavbeta5 through up-regulation of alphavbeta6 may protect SCCs from anoikis by activating an Akt survival signal.     

Basic FGF and Activin/Nodal but not LIF signaling sustain undifferentiated status of rabbit embryonic stem cells

Recently, we proposed that rabbit embryonic stem (ES) cells can be stable mammalian ES cells and can be a small animal model for human ES cell research. However, the signaling pathways controlling rabbit ES cell pluripotency remain largely unknown. Here we report that bFGF can maintain the undifferentiated status of rabbit ES cells and found that Activin/Nodal signaling through Smad2/3 activation is necessary to maintain the pluripotent status of rabbit ES cells. We further show that in spite of STAT3 in rabbit ES cells, LIF is dispensable for maintenance of undifferentiated status in rabbit ES cells. Although phosphorylation of Janus Kinase signal transducer and activator (JAK/STAT) disappeared after JAK-inhibitor treatment, OCT4 is constantly produced. When rabbit ES cells were cultured for more than 40 passages in the absence of LIF, expression of stem cell markers and teratoma formation were observed. Additionally, treatment with Rho-associated kinase (ROCK) inhibitor, Y27632, to rabbit ES cells significantly enhanced cell growth. These findings suggest that molecular mechanisms underlying rabbit ES cell self-renewal and pluripotency are similar to primate ES cells. Rabbit ES cells may provide a translational research model for the study of human diseases in vitro and applications to transplantation therapy.     

Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells

Human embryonic stem cells (hESCs) are routinely cultured on fibroblast feeder layers or in fibroblast-conditioned medium (CM). Bone morphogenetic proteins (BMPs) have previously been shown to induce hESC differentiation, in apparent contrast to mouse embryonic stem (ES) cells, in which BMP4 synergizes with leukemia inhibitory factor (LIF) to maintain self-renewal. Here we demonstrate that hESCs cultured in unconditioned medium (UM) are subjected to high levels of BMP signaling activity, which is reduced in CM. The BMP antagonist noggin synergizes with basic fibroblast growth factor (bFGF) to repress BMP signaling and sustain undifferentiated proliferation of hESCs in the absence of fibroblasts or CM. These findings suggest a basic difference in the self-renewal mechanism between mouse and human ES cells and simplify the culture of hESCs.     

Dynamic changes in histone H3 phosphoacetylation during early embryonic stem cell differentiation are directly mediated by mitogen- and stress-activated protein kinase 1 via activation of MAPK pathways

Embryonic stem (ES) cells are pluripotent cells capable of unlimited self-renewal and differentiation into the three embryonic germ layers under appropriate conditions. Mechanisms for control of the early period of differentiation, involving exit from the pluripotent state and lineage commitment, are not well understood. An emerging concept is that epigenetic histone modifications may play a role during this early period. We have found that upon differentiation of mouse ES cells by removal of the cytokine leukemia inhibitory factor, there is a global increase in coupled histone H3 phosphorylation (Ser-10)-acetylation (Lys-14) (H3 phosphoacetylation). We show that this occurs through activation of both the extracellular signal-regulated kinase (ERK) and p38 MAPK signaling pathways. Early ES cell differentiation is delayed using pharmacological inhibitors of the ERK and p38 pathways. One common point of convergence of these pathways is the activation of the mitogen- and stress-activated protein kinase 1 (MSK1). We show here that MSK1 is the critical mediator of differentiation-induced H3 phosphoacetylation using both the chemical inhibitor H89 and RNA interference. Interestingly, inhibition of H3 phosphoacetylation also alters gene expression during early differentiation. These results point to an important role for both epigenetic histone modifications and kinase pathways in modulating early ES differentiation.     

Human intestinal epithelial cell survival and anoikis. Differentiation state-distinct regulation and roles of protein kinase B/Akt isoforms

We have shown previously that human intestinal epithelial cell survival and anoikis are distinctively regulated according to the state of differentiation. Here we analyzed the roles of protein kinase B/Akt isoforms in such differentiation state distinctions. Anoikis was induced in undifferentiated and differentiated enterocytes by inhibition of focal adhesion kinase (Fak; pharmacologic inhibition or overexpression of dominant-negative mutants) or beta1 integrins (antibody blocking) or by maintaining cells in suspension. Expression/activation parameters of Akt isoforms (Akt-1, Akt-2, and Akt-3) and Fak were analyzed. Activity of Akt isoforms was also blocked by inhibition of phosphatidylinositol 3-kinase or by overexpression of dominant-negative mutants. Here we report the following. 1) The expression/activation levels of Akt-1 increase overall during enterocytic differentiation, and those of Akt-2 decrease, whereas Akt-3 is not expressed. 2) Akt-1 activation is dependent on beta1 integrins/Fak signaling, regardless of the differentiation state. 3) Akt-2 activation is dependent on beta1 integrins/Fak signaling in undifferentiated cells only. 4) Activation of Akt-1 is phosphatidylinositol 3-kinase-dependent, whereas that of Akt-2 is not. 5) Akt-2 does not promote survival or apoptosis/anoikis. 6) Akt-1 is essential for survival. 7) Akt-2 cannot substitute for Akt-1 in the suppression of anoikis. Hence, the expression and regulation of Akt isoforms show differentiation state-specific distinctions that ultimately reflect upon their selective implication in the mediation of human intestinal epithelial cell survival. These data provide new insights into the synchronized regulation of cell survival/death that is required in the dynamic renewal process of tissues such as the intestinal epithelium.     

Glutathione peroxidase-1 is required for self-renewal of murine embryonic stem cells

Embryonic stem (ES) cells are pluripotent cells that are capable of giving rise to any type of cells in the body and possess unlimited self-renewal potential. However, the exact regulatory mechanisms that govern the self-renewal ability of ES cells remain elusive. To understand the immediate early events during ES cell differentiation, we performed a proteomics study and analyzed the proteomic difference in murine ES cells before and after a 6-h spontaneous differentiation. We found that the expression level of glutathione peroxidase-1 (GPx-1), an antioxidant enzyme, is dramatically decreased upon the differentiation. Both knockdown of GPx-1 expression with shRNA and inhibiting GPx-1 activity by inhibitor led to the differentiation of ES cells. Furthermore, we showed that during early differentiation, the quick degradation of GPx-1 was mediated by proteasome. Thus, our data indicated that GPx-1 is a key regulator of self-renewal of murine embryonic stem cells.     

BCR-ABL induces opposite phenotypes in murine ES cells according to STAT3 activation levels

The mechanisms by which p210-BCR-ABL determines hematopoietic stem cells fate remain poorly understood. To better understand the behavior of BCR-ABL in pluripotent stem cells, we previously developed a murine embryonic stem (ES) cell model transformed by p210-BCR-ABL and reported that BCR-ABL activates STAT3, a major protein involved in ES cells self-renewal, which leads specifically to inhibition of ES cells differentiation. We show here that BCR-ABL either inhibits differentiation or, unexpectedly, induces a rapid commitment to differentiation of murine ES cells, according to the intracellular levels of activated STAT3. We show that inhibition of endogenous STAT3 activation with an inducible STAT3 protein with dominant-negative activity (STAT3F) results in an early, rapid and complete differentiation of BCR-ABL-expressing ES cells, whereas control ES cells retain a more undifferentiated phenotype. This phenomenon could be totally abrogated by PD98059, a specific MEK1 inhibitor, suggesting the involvement of mitogen-activated protein kinase (MAP-Kinase)/ERK1/2 pathway, which was found constitutively phosphorylated in BCR-ABL-expressing cells. In addition, BCR-ABL-expressing ES cells harboring low levels of activated STAT3 committed more rapidly through hematopoietic differentiation, since embryoid bodies (EBs) derived from these cells were able to generate numerous hematopoietic progenitors 2 days early. Moreover, BCR-ABL-expressing ES cells cultured first with low levels of activated STAT3 before EBs derivation displayed a more rapid loss of pluripotency than controls and failed to generate hematopoietic progenitors. This phenomenon was partially abrogated when ES cells were first exposed to PD98059 or to the tyrosine kinase inhibitor imatinib mesylate. From this predictive model, we suggest that variations of the activation levels in BCR-ABL substrates such as STAT3 may represent "instructive" secondary cooperating events involved in the transformation of the leukemic cell phenotype during the course of CML.     

Isolation of embryonic stem-like cells from equine blastocysts and their differentiation in vitro

Embryonic stem (ES) cells are pluripotent cells with the potential capacity to generate any type of cell. We describe here the isolation of pluripotent ES-like cells from equine blastocysts that have been frozen and thawed. Our two lines of ES-like cells (E-1 and E-2) appear to maintain a normal diploid karyotype indefinitely in culture in vitro and to express markers that are characteristic of ES cells from mice, namely, alkaline phosphatase, stage-specific embryonic antigen-1, STAT-3 and Oct 4. After culture of equine ES-like cells in vitro for more than 17 passages, some ES-like cells differentiated to neural precursor cells in the presence of basic fibroblast growth factor (bFGF), epidermal growth factor and platelet-derived growth factor. We also developed a protocol that resulted in the differentiation of ES-like cells in vitro to hematopoietic and endothelial cell lineages in response to bFGF, stem cell factor and oncostatin M. Our observations set the stage for future developments that may allow the use of equine ES-like cells for the treatment of neurological and hematopoietic disorders.     

Potential of embryonic stem cells

Embryonic stem (ES) cells are pluripotent cell lines established from undifferentiated embryonic cells characterized by nearly unlimited self-renewal and differentiation capacity. During differentiation in vitro, ES cells were found to be able to develop into specialized somatic cells types and to recapitulate processes of early embryonic development. These properties allow to use ES cells as model system for studying early embryonic development by gain- or loss-of-function approaches, or to investigate the effects of drugs and environmental factors on differentiation and cell function in embryotoxicity and pharmacology. Now, ES cells derived of human blastocysts may be used for the generation of somatic precursor or differentiated cells in cell and tissue therapy. The review presents data of mouse ES cell differentiation and gives an outlook on future perspectives and problems of using human ES cells in regenerative medicine.     

Bim is an apoptosis sensor that responds to loss of survival signals delivered by epidermal growth factor but not those provided by integrins

Anoikis is a rapid apoptosis response that is initiated within a few minutes after inhibition of integrin signaling. In mammary epithelia, anoikis is mediated by subcellular translocation of Bax from the cytosol to mitochondria where it activates the intrinsic apoptosis pathway. The Bcl-2 homology 3 domain-only protein, Bim, has been proposed to have a key role in the apoptosis response of an epithelial cell line with reduced sensitivity to loss of integrin signaling, which undergoes apoptosis over a period of several days in suspension culture. Here we tested the involvement of Bim in the rapid anoikis response of mouse mammary epithelial cells and discovered that Bim does not have a role in detecting integrin-mediated signals. Instead Bim senses the loss of survival cues mediated by epidermal growth factor. Cell lines selected over many passages in culture have lost much of their sensitivity to anoikis signals arising from an altered cellular microenvironment and may undergo apoptosis through acquired mechanisms.